I moved to Florida a number of months ago, but I am only now starting a cryonics group in the Broward County, Florida area. As a first event I have booked a table at the Peking Tokyo Buffet restaurant on 1219 South Federal Hwy, Deerfield Beach, Florida for dinner in the early evening (7 P.M. to 9 P.M.) on Thursday, January 24th. All those interested in cryonics in Broward county and adjoining areas are invited to attend.

This will be the first meeting of this group so the main goal will be to meet other cryonicists or cryonics-interested people in the South Florida area. All-you-can-eat buffet with a wide selection of foods for only $10.95, but no purchase or meal is required for those who simply want to socialize and discuss cryonics while others eat. Drop-in any time between 7 P.M. and 9 P.M., but closer to 7 P.M. would be preferred. Use exit 41 from I95, drive East along SW (becomes SE at Dixie) 10th Street, and turn right on Federal Highway (US Route 1) to reach the Peking Tokyo Buffet in Deerfield Beach. Anyone interested in cryonics is welcome to attend.

I have been phoning cryonicists in the area and quite a few seem certain to attend. I have also started groups on Facebook and Meetup for this purpose, which I invite others to join, but don’t get the wrong impression that the buffet event on Thursday, January 24th is going to be as poorly attended as the Facebook or Meetup groups might make you imagine.

Some observers believe that cryonics advocates are reluctant to subject their theories to experimental scrutiny because this could damage their (uncritical) belief in future resuscitation. Similarly, one might think that cryonicists would react with a mix of hostility and dismissal to alternative strategies for personal survival. Nothing could be further from the truth. In fact, it is exactly because our personal survival is at stake that forces us to be wary of dogmatism.

For this reason, I have always been interested in chemical fixation as a (low cost) alternative for cryonics. In fact, years before all the talk about the “connectome” and “plastination” I spent considerable time exchanging messages with Michael Perry at Alcor about the technical and practical feasibility of chemical brain preservation. But no matter how open minded I tried to be about this approach, I kept running into the same challenges over and over again.

The challenge that has concerned me the most is whether a delayed start of chemical brain fixation will produce incomplete distribution of the chemical fixative in the brain because of ischemia-induced perfusion impairment. Thinking about the technical problem of “no-reflow” is not the first thing on the mind of someone who first hears about the idea of using chemical fixatives to preserve the brain. In my case, this concern was not just “theoretical.” In my lab I have spent many years looking at the effects of cerebral ischemia on cryopreservation and chemical fixation. Last year we decided to broaden our investigations to delayed chemical fixation and we have not been pleased at what we have observed so far. After 1.5 years of room temperature storage the delayed aldehyde fixed brains are falling apart and continue to decompose. In small animals one might imagine that such perfusion impairment could be overcome by immersing the brains in the fixative instead but human brains are simply too large. By the time that the fixative would have reached the core of the brain, extensive autolysis will have occurred.

Another complex problem is to identify a fixation and polymerization protocol that fixes all identity-critical parts of the brain. If aldehydes do not completely fix the lipids in the brain, should we add strong oxidizing heavy metals to stabilize lipids? This is possible in theory but, as a general rule, these chemicals are either very expensive or dangerous to use (or both). Even if we are able to identify a chemical fixation protocol for the brain that can do the job, how can we know that such brains are stable for very long periods of time? Should we follow fixation by embedding with a polymer to inhibit residual biochemical activity? To my knowledge, there is no known embedding protocol that is scalable to human brains due to the extreme viscosity of these plastics.

Recently these issues took a more personal nature for me when I had to think really hard about a reasonable but affordable longterm preservation protocol for a companion animal. I spent many days reading the electron microscopy and fixation literature to come up with a protocol that was better than aldehyde fixation and low temperature storage. Adding calcium to the fixative? What about phenol? Post-fixation perfusion of a viscous cryoprotectant to allow storage at subzero temperatures? That is when I really started appreciating the “magic” of cold temperatures.

Absent a vitrification agent, cryogenic temperatures can cause extensive damage to cells. But one thing we know: whatever the nature of this damage, as soon the brain is below the glass transition temperature of -130°C, all water is either frozen or a vitrified rigid solid. We do not have to worry about any damage getting worse over time, or whether some biomolecules have not been fixed. Cold may be “crude” in its effects but it is exactly because no biochemical process can escape inhibition at very low temperatures that makes it such a powerful personal survival technology.

Originally published as a column (Quod incepimus conficiemus) in Cryonics magazine, January, 2013

In her book Reconstructing Illness: Studies in Pathography, Anne Hunsaker Hawkins proposes that the modern pathography is replacing the accounts of religious conversion that were popular in earlier eras. What is a pathography? One definition that I found is “the study of the life of an individual or the history of a community with regard to the influence of a particular disease or psychological disorder.” Reconstructing Illness is an extensive study of this genre, how individuals deal with a diagnosis of a serious illness, and its broader role for medical caregivers and society.

One thing that I was wondering about while reading this is whether there are any pathographies of aging. There is no shortage of pathographies about cancer, HIV/AIDS, dementia (etc.) but I was curious if anyone had ever considered writing about the individual experience of the aging process and its inevitable outcome, death. Hawkins’s book has a very useful list of pathographies organized by disease. Perusing this list provides one with a good understanding of which kind of pathographies are popular but I failed to find even one title that explicitly concerns aging. Similarly, a search on “pathography of aging” on the internet did not produce any results. Sure, there are many books about facing death (or dealing with the death of a loved one) or the challenges and opportunities associated with growing older. But I am not aware of any account that treats the aging process in a format that is remotely similar to the descriptions of disease we meet in the pathography, let alone one where the aging process is described as a battle to be undertaken.

This should not be surprising. For most of us, disease is an abnormal condition that is defined relative to the normal aging process. Although a lot of disease is closely associated with aging, most people hesitate to call the aging process itself a disease because it would render the conventional use of the word disease problematic. There are diseases that are characterized by rapid aging in children, such as progeria, but we do call such conditions a disease because the pace at which these children grow older is not normal. In fact, pathographies of accelerated aging diseases might be the closest thing that approaches a pathography of aging.

Regardless of one’s perspective on the causes or mechanisms of aging, if we look at aging at the molecular level we will find a progressive accumulation of damage as we grow older. Whatever we mean by “aging gracefully,” this accumulation of damage stops for no one and ultimately results in death. Because aging is normal, and no one is being diagnosed with aging, there is not a clear, identifiable, moment in life that triggers the experience and events that are documented in the typical pathography. In fact, the universal nature of human aging and our propensity to react more strongly to unexpected events strongly biases humans to respond to specific diseases and not the aging process itself. What we seem to care about is abnormal deterioration and death, not the deterioration and death that is universal and foreseeable.

Not all people react in such a passive manner to aging. Not anymore. To some of us the relatively slow pace of physiological deterioration is a source of anxiety and the fact that it is a universal phenomenon does not provide solace, especially when medical technologies to halt or reverse aging can be envisioned and pursued. What sets humans apart from other animals is that we can recognize a universal condition and not be satisfied with it. Aging is an undeniable source of suffering and loss of dignity, sets the stage for separation and death, and favors short-term thinking over long-term responsibilities. It will only be a matter of time before the first pathographies of those who succumbed to the process while consciously fighting it will reach us.

Originally published as a column (Quod incepimus conficiemus) in Cryonics magazine 2012-6

A common complaint against cryonics is that existing cryopreservation technologies may not be good enough to preserve the ultrastructure of the human brain. Advocates of cryonics often object that such views do not reflect actual inspection of the evidence of cryopreserved brains but instead reflect misconceptions about “freezing” and ice formation rupturing cells. But the more fundamental misconception rests on the view that for cryonics to work flawless preservation of the brain is absolutely essential.

This view is not only mistaken but holds cryonics to higher standards than those applied in conventional medicine. In medicine it is routine for patients to present themselves with conditions in which an organ or tissue has been changed from its normal condition (or appearance) as a result of disease or trauma. Restoring normal structure or function is the essence of most medical treatments.

One might object that in the case of cryonics we are concerned with the brain, which distinguishes itself from other organs that encodes highly individual information. If a portion of the brain is erased we cannot consult another brain or medical textbook to infer its original state. We can admit that this is a valid observation but it is not necessarily a fatal argument against cryonics, provided the damage has not reached the point of complete destruction or indecipherability.

There is a difference between damage and obliteration. If we look at electron micrographs of brain tissue produced at various points in time after circulatory arrest (“death”) we will observe progressive alterations of synapses, cell membranes, organelles etc. We describe such changes with a mental (or actual) map of how they normally look like in mind. At this level the fact that the brain is a highly individual organ is no longer relevant because we know the universal biochemical language in which this identity is written. At this point the real question becomes at which point is it not even possible to infer the original condition of the brain. As far as we understand this today, this may be a question of many hours, if not days.

This robustness of identity-critical information in the brain may seem to contradict the routine observation in emergency medicine that there is a much narrower time limit for successful resuscitation from cardiac arrest. The crucial difference here is that we are no longer talking about the ability to infer identity-critical information but restore physiological function. But function is a lot more vulnerable to metabolic and biochemical changes than the wiring of the brain. In fact, if function were a necessary requirement to infer information a lot of existing forensic and archeological science would be impossible.

In approaching cryonics it is important to recognize the distinction between preserving and inferring. In this way we can better assess the prospects for resuscitating patients who were cryopreserved under nonideal conditions and/or with older technologies.

Originally published as a column (Quod incepimus conficiemus) in Cryonics magazine 2012-5.

As we learn more about the human genome, there will be an increasing recognition that general diet recommendations are going to give way to diet recommendations that more closely track the genotype of individuals. For those interested in healthy life extension an important question concerns the relationship between ApoE status and diet. In Why We Age : What Science Is Discovering About the Body’s Journey Through Life (1997) Steven N. Austad writes:

.. piles of evidence suggest that certain genes have a major impact on the development of atherosclerosis, probably the major disease of aging in the Western world. One of those genes is the Apolipoprotein E, usually abbreviated ApoE, which is involved in processing dietary fat. People with one form of the gene, called e4, have higher blood cholesterol (as well as higher LDL, or ”bad” cholesterol) levels than people with other forms of the gene. Finns have the highest rate of atherosclerosis in the world and also have one of the world’s highest frequencies of e4. The Japanese have the world’s lowest national rate of atherosclerosis and also among the world’s lowest frequency of e4. So you could call e4 an atherosclerosis gene. But this would be misleading, because the world’s highest frequency of e4 is found in a country, Papua New Guinea, where until recently atherosclerosis was virtually unknown.

People living in the bush in Papua New Guinea eat a low-fat diet (less than 5 percent fat, compared with 30 to 40 percent fat in an American diet) from necessity rather than choice. Their daily life also involves exercise at levels that would cripple or kill most Americans, even the athletically inclined….So genes operate not in a vacuum but in a specific environment. This is something to bear in mind when reading of the discovery of new “longevity” genes. For instance, there is another form of the ApoE gene, e2, which appears to lower blood cholesterol and therefore probably protects against developing atherosclerosis. Is this a longevity gene? It depends on the environment. Where people eat a lot of fat and don’t exercise, it may well be a longevity gene. In fact, French centenarians are about twice as likely to have this gene as the French population as a whole. But in other environments, the gene may well have little or no effect.

What these examples suggest, besides the difficulty in defining genes with respect to longevity, is that unless we understand how a particular gene is influenced by a particular environment, it will be difficult to translate the effects of genes from animals to humans. This is why most gerontologists are hesitant to claim too much about the relevance to humans of genes now being found with increasing frequency in simple organisms such as fungi and worms that seem to slow aging dramatically. It is difficult to draw parallels between human and worm and fungal environments. (p-41-43)

It is important to keep this point in mind when one considers the pro- and cons of a popular diet. For example, the Paleo Diet has become increasingly popular in the life extension & transhumanist communities. But if the observations of Austad are correct, a diet high in (saturated) fat could have adverse consequences for carriers of one or two copies of the ApoE4 gene. In fact, in her book The Perfect Gene Diet Pamela McDonald steers ApoE4 carriers in the direction of a vegetarian / vegan diet. As we learn more about the ideal diet for carriers of the ApoE4 gene, further refinements may be expected.

Another interesting emerging finding about ApoE4 is that its effect on having a higher probability of developing late-onset Alzheimer’s disease may be dependent on gender. A number of preliminary studies have found that the risk for developing Alzheimer’s disease for males with just one copy of the ApoE4 gene may not be much different from that of individuals who carry the more common ApoE3 gene:

Together with the previous meta-analysis, the data support the idea that a man with one E4 allele may not have much more risk of AD than an E3 homozygote, Greicius said, but added, “If you have two copies of the E4 allele, whether you are a man or a woman, there is no question that your risk leaps tremendously.” He is analyzing older datasets to see if the interaction between gender and ApoE genotype holds, and is also looking for genes that act synergistically with ApoE in women but not men.

If there is anything that is becoming clear from such studies it is that it will be increasingly inadequate to make sweeping statements about lifestyle, diet, and longevity without taking into account ethnicity, gender, age, genotype, and environment of a person. This does not mean that all general recommendations should be discarded. For example, there could be good reasons to believe that a low calorie diet and (moderate) exercise benefit most people. But when it is comes to the nitty gritty of what to eat and how to exercise a more personalized approach is warranted.

The recent symposium on cryonics and brain-threatening disorders was a major success. On Saturday, July 7, 2012, around 30 people attended the first ever symposium on dementia and cryonics in Portland, Oregon. The symposium started with a brief introduction by Institute for Evidence Based Cryonics President Aschwin de Wolf, who emphasized why people with cryonics arrangements have a clear interest in understanding and avoiding dementia. The first speaker, Chana de Wolf, introduced the audience to the topic of adult neurogenesis, the two areas in the brain where it occurs, and how little we still understand about it. Aubrey de Grey then talked about the SENS approach to rejuvenation and how some emerging damage repair bio-technologies might be able to also reverse neurodegenerative diseases such as Alzheimer’s disease. Cryonics Institute President Ben Best followed Aubrey’s presentation with a technical introduction about the pathophysiology of Alzheimer’s disease and the treatments that are currently being investigated. Ben is maintaining a page about the molecular mechanisms of Alzheimer’s disease on his personal website.

After the break Alcor staff member Mike Perry presented a detailed analysis of a recent paper in which cerebrospinal fluid samples could predict the onset of Alzheimer’s diseases many years before the first signs of cognitive impairment, a finding that holds great promise for life-extensionists, and those with an increased risk for Alzheimer’s disease in particular. Institute for Evidence Based Cryonics Board member Keegan Macintosh then presented a rigorous legal analysis of the Thomas Donaldson case and indicated how the case could have been argued more persuasively then and now. The last speaker of the day was Alcor President Max More who introduced the concept of the extended mind and its relevance to cryonics and neurodegenerative diseases, which prompted a useful exchange about the desirability of cryonics organizations facilitating members to store identity-critical information. The official meeting ended with a panel discussion moderated by Aschwin de Wolf in which all the speakers took questions from the audience and other speakers.

The program and panel left ample time for interaction between speakers and the audience. The topic of avoiding dementia and what to do when a cryonicist is diagnosed with a brain threatening disorder received a lot of attention. Despite the rather disturbing subject of the symposium there seemed to be a general recognition that it was extremely valuable to explore this topic in the context of cryonics. Some suggestions of how to deal with dementia were made that had not been previously discussed in cryonics publications.

It is not likely that we will organize a symposium about this topic every year but there was a strong interest in organizing meetings about other topics on a regular basis in the Pacific Northwest.

The slides of all but one of the presenters are available on the symposium page and a video recording of Aubrey de Grey’s talk was made by one person in the audience. A more detailed report of the symposium will appear in an upcoming issue of Alcor’s Cryonics magazine.

In a few days the Institute for Evidence Based Cryonics and Cryonics NW will host a symposium on Cryonics and Brain-Threatening Disorders. We care deeply about this issue and some of us have observed fellow cryonicists succumb to (advanced) dementia prior to their cryopreservation – or worse, the disease compromised their understanding of their cryonics arrangements and what it takes to keep them in place. Still, we have also observed that many cryonicists stick their head in the sand about this topic or are (excessively) optimistic that their diet or lifestyle will save them from such a fate. We would like to share this optimism but we think this line of thinking is neither realistic nor rational. In addition, dementia is not not necessarily an outcome of genes or age but can occur as a result of other diseases and accidents. We may feel comfortable about contemporary cryopreservation technologies but there is no clear answer to acute or progressive destruction of the brain. As Mike Darwin has pointed out, there is an urgent need for a “Brain Centered Approach to Geroprotection for Cryonicists.”

To facilitate more exchange between the presenters and the audience we have added a panel about cryonics and dementia at the end of the day. We have no illusions about being able to solve this problem soon but a better understanding of early diagnosis, available treatments, foreseeable interventions, and legal options for cryonicists is an important first step.

Attending with Argentine biogerontologist and Cryonics Institute Member Rudy Goya may have reduced the interaction I had with the cryobiologists. There were fewer sessions than usual, and thus more free time. The welcome reception was not held until the evening of the first day of the sessions.

The first session dealt with an aspect of Argentine cultural heritage, the Llullaillaco children — three Inca children who had been mummified by dehydration high on a volcano and preserved for over 500 years. Two of children were selected by the Incas because they were “perfect” (beautiful and pure) at 6 or 7 years of age. It was believe to be an honor to go directly to heaven, not really death or sacrifice. The children were given an intoxicant and buried alive atop the Llullaillaco volcano. Much of the session focused on the conditions that caused the children to be so well-preserved, and the conditions the curators should use to preserve the children for the future — involving careful regulation of temperature, atmosphere, humidity, and an environment inhospitable to most microbes.

If reanimated cryonicists receive anything like the care these children are receiving, there should be no concerns about being welcome in the future. In a sense, the Incas had it right when thinking they were sending the children to heaven. Of course the Inca children were deprived of life and are unable to experience or enjoy their treatment by modern curators — and cryonicists should not encourage hastening death based on reliance on unproven future technologies.

At this conference there were special “How to do it?” sessions overlapping part of the lunch hour that focused on practical techniques unrelated to the experimental results and theoretical considerations covered in the regular sessions. Sunday’s topic was proteomic analysis, which covered removal, isolation, and identification of proteins from cells. The presenter (from the Institute of Molecular Cell Biology in Rosario) claimed that instrumentation allowing high throughput and resolution had given proteomics a maturity comparable to genomics.

Adam Higgins reported on an improved procedure for washing glycerol from red blood cells. Currently about 99% of banked blood is stored at refrigerator temperature (2-4ºC), with a shelf life of 42 days. Only 1% of blood (mostly rare blood types) is cryopreserved with glycerol and stored at −80ºC, with a shelf life of ten years. A major deterrent preventing more blood from being banked at −80ºC is the 30-60 minute glycerol washout procedure. Adam’s group developed a procedure that can wash the glycerol out in 30 seconds, but 5 seconds longer or shorter results in too much hemolysis. A three minute washout procedure is less time sensitive (one minute longer or shorter is tolerable), but the method needs to be scaled-up from the 0.5 milliliter test volumes being used.

On Monday, Peter Mazur reported that in vitrifying mouse oocytes, it is the warming rate and not the cooling rate that is most critical for success. He spoke of microwave warming and the problem of thermal runaway (uneven warming). Ice blockers would not cross cell membranes, and thus would not be of use against intracellular ice formation. Pier Morin reported on miRNA microarray assessment of miRNA expression of the freeze-tolerant insect goldenrod gall fly at control (+5ºC) and freezing (−15ºC) temperatures. mIR-210 was down-regulated and mIR-1 was up-regulated at freezing temperature (the latter is involved in cell cycle regulation).

Ali Eroglu reported on epigenetic perturbation resulting from human oocyte cryopreservation techniques. Both the slow freezing and vitrification methods he used resulted in down-regulated expression of H19 and Ube3a genes. Igf2r was down-regulated by vitrification, but not by slow freezing.

Monday’s “How to do it?” session described a combination of nanotechnology and stem cells for tissue engineering. Specifically, electrospinning can be used to create a nanometer scale web of biodegradable fibers that can be populated with mesenchymal stem cells by electrospraying. The main challenge is vascularization of the tissue. Vascular Endothelial Growth Factor (VEGF) increases cell adhesion, but not necessarily vascularization.

Barry Fuller reported on successful hypothermic perfusion of liver. A kidney hypothermic perfusion machine has been in operation for ten years, but liver has been more challenging, because of its large size and the fact that two vessels supply the organ (hepatic artery and portal vein). The liver hypothermia perfusion machine uses two pumps.

PhD student Na Guan described her study of gene expression changes associated with chilling injury of rat liver slices. Cryoprotectant solutions supplied by 21CM (Greg Fahy) were used to ensure no ice formation interfered with the process. ATP levels indicated that the cryoprotectant solutions used were causing no damage, although the composition of those solutions was not disclosed. 1108 genes were observed, of which 251 were up-regulated and 77 were down-regulated by chilling at −15ºC. Focusing on the top ten up- and down-regulated genes: inflammatory and DNA repair genes were considerably up-regulated, and genes associated with biosynthesis of cholesterol and polyunsaturated fatty acids were down-regulated. The latter seems paradoxical in light of the up-regulation of cell surface-linked signaling pathways, which indicate cell membrane injury.

During the question period, both Andreas Sputtek and Arthur Rowe were sharply critical of the undisclosed composition of the 21CM cryoprotectant solutions being used. Sputtek said that because science is about disclosure of methods and materials, Guan’s work was not science. Guan said she had begged 21CM for disclosure, but said she was told that anyone wanting to replicate the experiments could buy the solutions from 21CM. Tiantian Zhang said that gene analysis only done 30 minutes after chilling injury does not give the whole picture. She said that in her own work doing gene analysis of fish oocytes or embryoes after chilling injury, gene expression changes dramatically with time — that it is a mistake to only analyze the expression 30 minutes after exposure as Guan had done. After the presentation, Arthur Rowe spoke with Guan telling her how much trouble he has had over the years with her collaborator (Dr. Fahy) in connection with the non-disclosure issue. I spoke with Guan myself after her presentation. She told me that the greatest chilling injury occurs at −90ºC. She also said that she would be getting her PhD in July and did not know who would be continuing her work. When I spoke to Dr. Fahy about the presentation, he told me that the composition of the vitrification solution had been disclosed and that Guan was mistaken in believing that she could not disclose the composition.

Tuesday morning had been scheduled to begin with a lecture by Ken Storey. Storey typically has no interest in what other cryobiologists have to say, is fairly ignorant of areas of cryobiology outside of hibernation and effects of low temperature on animals in nature, and only comes for his own presentation before leaving. His ignorance is on display when journalists get him to do cryonics-bashing, which he does with relish, but the general public only sees the comments of a respected cryobiologist, not the ignorant misunderstandings of cryobiology. I would not have expected Storey to come all the way to Rosario, Argentina only for his own presentation, but this is what he attempted to do — and he missed one of his flight connections. Ironically, this year Storey was honored by being made a Fellow in the Society for Cryobiology. Storey does, admittedly, have a fabulous knowledge of molecular biology, and is an outstanding scientist in connection with his own work.

To compensate for Storey’s absence the conference organizers arranged a makeshift follow-up session on the Llullaillaco children. This wasn’t entirely a waste, because many issues had not been addressed in the first round. I was going to question using a 2% oxygen and 98% nitrogen atmosphere for the children rather than pure nitrogen, but Barry Fuller raised this objection before I was called upon. I did, nonetheless, suggest that the goal should be to perfect the preservation environment rather than try to recreate the conditions of the mountain. Even this had not been done because the relative humidity had been raised to 70% on the bad advice of an expert rather than held to the 40% present on the volcano. The children were reportedly gaining 300 grams per year, probably from the humidity. There is a lower humidity limit below which no microorganisms can grow, but 0% relative humidity in the −20ºC preservation chambers would run the risk of freeze-drying.

For the second session on Tuesday, John Crowe had been scheduled to lead a symposium composed of 3 other speakers besides himself, but all of the other 3 speakers cancelled-out. John, nonetheless, did an excellent job of speaking for the whole session on the basis of his own work. John is an expert in dehydration and freeze-drying of organisms as well as on tardigrades and trehalose. Drying DNA with trehalose prevents fragmentation, and drying proteins with trehalose prevents denaturation. John discovered that drying liposomes with trehalose prevents membrane fusion — although he lost most of the patent rights on commercially valuable processes by publishing too soon. Dehydration of samples containing sucrose drives the glass transition temperature (Tg) from 20ºC to 60ºC, but dehydration of samples containing trehalose raises the Tg from 20ºC to 120ºC. More recently, however, it has been found that LEA proteins can be as protective as trehalose, but in a way that is distinctive and complementary to trehalose — stopping liposome fusion, preventing protein aggregation, and changing sample Tg. Yeast cells are protected against dehydration damage not only by trehalose, but by the trehalose transporter protein which exports the trehalose to the exterior membrane surface and imports the trehalose to the internal membranes of organelles such as mitochondria. But although the genome of tardigrades has been sequenced, the tardigrade trehalose transporter has not yet been identified.

Barbara Reed is probably the world’s foremost expert on plant cryopreservation, and she has spoken a lot about the benefits of antioxidants for cryoprotection. But the presentation Barbara gave on Wednesday gave me the strongest indication that oxidative stress could be a significant mechanism of cryoprotectant toxicity. Not only because a variety of cryopreserved plants show improved viability with Vitamin E, Vitamin C (if iron is removed), lipoic acid, glutathione, and melatonin — but because oxidative damage was shown to increase significantly associated with cryoprotectant loading.

Roland Fleck works with the UK Stem Cell Bank. The Bank conducted studies indicating that a 2-step freezing protocol results in better viability than vitrification. But examining the results of 8 technicians showed that in the hands of the most experienced technician vitrification was as effective as the 2-step freezing protocol. Protocols should not be so highly dependent upon technician expertise. After his presentation, Roland told me he was concerned that he was only able to assay viability by the use of trypan blue, which only indicates membrane integrity and does not provide a very fine measure of cell function. He said that the requirement to use the trypan blue viability assay was imposed by bureaucrats or scientists who do not have much knowledge of cryobiology.

Igor Katkov said that he believes any sperm cell can be vitrified simply by choosing the right cooling and warming rate. He said he was advised by his patent attorney to drop seven slides from his PowerPoint presentation.

At the business meeting the Society membership was reported to be down to 186. The journal CRYOBIOLOGY continues to be profitable. CRYOBIOLOGY has a 33% rejection rate, a 1.83 impact factor, and 33 Members on the Editorial Board. The Society has $300,000, which the IRS thinks is too much for a charitable organization, but the IRS is allowing the society to retain tax-exempt status. Increasing travel awards is the preferred use of money, but there is a problem that on the one hand travel awards are a taxable benefit, and on the other hand it is illegal to pay the taxes on travel awards. The 2013 conference (the 50th annual conference) is to be held in Washington, DC, where the first conferences were held. The 2014 conference might be Istanbul, Turkey and the 2015 conference could be in Isreal, but definite decisions have not been made.

Last year’s new Society for Cryobiology Fellows Barbara Reed and John Crowe each gave presentations reviewing their careers. Barbara Reed began as a plant biologist in 1985, but was brought into the field of cryobiology by a need to preserve germ tissue. John Crowe said that after the Sputnik shock of 1957 the US government sought to encourage more young people to go into science, including him. As a teenager, John was sent to a number of different science laboratories on his summer vacations. John considers himself more of a “dryobiologist” than a cryobiologist. He entertained us with photos taken in the many exotic countries he and and his wife have visited since his retirement.

The two new Fellows for 2013 are Ken Storey and Mehmet Toner.

This conference was attended by not more than about 80 people, at least half of whom were South America. There were maybe 30 or so hard-core Society for Cryobiology Members. This was my 9th annual meeting in a row, but for the most part I made little effort to relate to the cryobiologists, although one of my intentions in attending these meetings has been to soften the hostility of cryobiologists to cryonicists. I sat near the front of the meetings with Rudy who told me that he learned a great deal about the cryobiology behind cryonics practices by attending this conference. Very many of the cryobiologists were reporting on using vitrification at this conference, and including articular cartilage and plant tissue as well as single cells. I was fairly active in my questioning and comments — about which a few of the cryobiologists complimented me.

I lost my sense of urgency about talking to Peter Mazur. Peter recently told a journalist that although it is not possible to prove that the chance of cryonics patients being reanimated are zero, “you can, I think demonstrated that the probability of its being done is so extremely low that effectively it is zero” [CANADIAN MEDICAL ASSOCIATION JOURNAL; Monette, M; The Church of Cryopreservation; 184(7):749 (2012)] I am curious about the demonstration Peter has in mind, but I am also committed to learning from cryobiologists rather than arguing with them about cryonics. Peter walked away a few years ago when I asked him when solution effects rather than mechanical damage cause injury to cells due to slow cooling, so that may be a touchy subject with Peter as well.

I did, however, pepper John Crowe with questions — finding him to be friendly and informative. John confirmed what Peter Mazur had told me about cells being able to tolerate the loss of all osmotic water (freezable water, which constitutes at least 80% of cell water) without injury — a matter of great relevance in the vitrification of cryonics patients (assuming inter-cellular effects are not of great significance).

I sought-out Ali Eroglu, with whom I have had little interaction in the past, calling his attention to an article in the most recent issue of CRYOBIOLOGY about transfection of mammalian ovary cells with trehalose [CRYOBIOLOGY; Chakraborty,N; 64(2):91-96 (2012)]. Ali has microinjected oocytes with trehalose (along with low concentrations of DMSO to protect the mitochondria) [BIOLOGY OF REPRODUCTION; Eroglu,A; 80(1):70-78 (2009)]. Ali had not seen the CRYOBIOLOGY article, but he told me that ovarian tissue is easier to work with than oocytes.

At the final banquet I sat next to one of the conference organizers. He told me that John G Baust had been supposed to conduct a symposium, but had cancelled the whole thing a month before the conference without giving any explanation. He agreed with the comments I had made about the Llullaillaco children, and told me that a committee of cryobiologists was going to supplement the questionable advice that the Argentine government has been getting from a single advisor in New York. He told me that National Geographic had discovered the children and attempted to remove them from Argentina on a midnight flight, but the Argentine government got wind of the plan and intervened. Nonetheless, the children were simply kept in −20ºC freezers for several years while planning and building better preservation chambers.

The return bus trip to BA on Thursday took the entire afternoon — much longer than I would have expected. I sat next to Adam Higgins on the bus, and spoke with him much of the time, mostly about his life and work, as well as about our experiences in Argentina. Adam knew Spanish fairly well because he has spent four months of language immersion living in Equador (and visiting the Galapagos Islands). If he gets a patent for deglycerolizing blood, the University would get half the royalties and he would split his half with his collaborators. The advantages of his method would be the ten year rather than 42-day shelf life for banked blood, and the greatly reduced washout time. The latter is a significant savings in labor costs, but would have to be weighed against greater electrical costs for a −80ºC freezer as opposed to refrigeration. Even if he is successful in perfecting his methods, he thinks that the blood banking industry is too conservative to be captivated by superior storage methods. Adam has attended most of the annual conferences since I began attending in 2004, and told me that he would like to become a Governor of the Society. Not once did Adam ask me what work I do, and he evidently does not know because he was surprised when I told him I am not a Member of the Society for Cryobiology. Whether or not I am formally accepted as a Member, my attendance at these conferences is implanting me into the consciousness of the cryobiologists as being a member of their community.

It is generally not the task of scientists to consider the legal, financial, and logistical limitations when searching for biomedical breakthroughs but there are good examples where considering the real-world applications of a technology can be instructive. Research aimed at preservation of brains (or the “connectome”) is such an example. Even if chemopreservation can be demonstrated to preserve the intricate wiring of the brain, it can be safely assumed that there will not be a massive change in demand for brain preservation technologies (especially if the technology is too strongly tied to mind uploading). As a consequence, providers of chemopreservation will most likely operate in the same environment as providers of cryonics. That means that, as a general rule, there will be a delay between pronouncement of legal death and the start of procedures.

There is now more than 40 years of mainstream biomedical research demonstrating that even short interruptions of circulation (under normothermic conditions) can produce perfusion impairment in the brain. As has been demonstrated by cryonics researcher Mike Darwin and my own lab, Advanced Neural Biosciences, this “no-reflow” can produce poor distribution of cryoprotectants (including vitrification agents) and associated freezing. One serious concern that cryonics researchers have about chemopreservation-in-the-real-world is that poorly chemically fixed brains will be prone to autolysis during long-term storage. This limitation of chemopreservation applies to both “conventional” biological resuscitation scenarios as to whole brain emulation. One can only recover (or “upload”) what is preserved – or can be inferred. And as far as we understand things today, the advantage of temperature as a long-term preservation method is that it does not depend on a healthy, non-ischemic circulatory system. Cryopreservation of an ischemic brain can produce ice formation, but as soon as it is placed in liquid nitrogen, cold will “fix” whatever there is without further degradation. The same thing cannot be said about chemopreservation under poor conditions.

There is an understandable tendency to compare brain preservation protocols under ideal conditions and favor the method that produces the best preservation. But support for either technology cannot be solely based on results produces under controlled lab conditions. Personal survival technologies should be evaluated under conditions that are most likely to be encountered by organizations that will offer them. Demonstrating that chemical fixation (and plastination) can preserve the connectome is a laudable goal but the case for chemopreservation as a clinical experimental preservation method requires a persuasive response to the objection that delays in fixation can frustrate the aims of chemopreservation in the most fundamental manner.

One interesting aspect of the cryonics vs chemopreservation debate, though, is that it appears that some people simply feel more comfortable with one of the approaches. People who have shown the slightest interest in human cryopreservation can get really excited about the idea of chemical brain preservation. This indicates that if both approaches would be pursued actively, the growth of chemopreservation would not necessarily be at the expense of cryonics but there would be a growth in the total number of people making bio-preservation arrangements aimed at personal survival. But as Mike Darwin has recently pointed out, chemopreservation is not at the stage where it can be responsibly offered. The growth of this field requires a committed group of individuals who will research, develop, and implement this program. Chemopreservation does not need to be perfected before being offered (neither was cryonics) but so far most advocacy has been mostly at the conceptual level.

The schedule for the upcoming Portland Cryonics and Brain-Threatening Disorders Symposium has been published.

On July 7, 2012 a number of high-profile and upcoming speakers in the cryonics and life extension community will talk about identity-destroying brain disorders and how diseases like Alzheimer’s can frustrate the objectives of the most ambitious life extentionists. Topics that will be discussed include the pathophysiology of Alzheimer’s, emerging early-diagnosis technologies, neurogenesis in adults, repairing the aging brain, and legal options for people diagnosed with a brain-destroying disease.